Identification of protein/protein interactions is at the core of understanding the biological processes occurring in living cells. Traditionally, the potential interacting proteins have been identified by genetic methods (two hybrid screens) with subsequent verification of the interaction by co-immunoprecipitation. While this method has been very successful for detection of two interacting proteins, it is of limited utility when more complex protein aggregates such as ribosomes, splice complexes or transcription complexes are investigated.
To identify and isolate yeast complex protein aggregates, an alternative method has been developed by Seraphin et al. (Rigaut et al., 1999, Nature Biotech., 17:1030-1032; Puig et al., 2001, Methods, 24: 218-219; U.S. 2002/0061513, reviewed in Terpe et al., 2003, App. Microbiol. Biotechnol., 60:523-533). This method combines purification of the protein complex of interest using two different affinity purification tags fused to at least one known protein component of a complex of interest by genetic methods, with subsequent mass spectroscopy to identify the unknown components of the isolated complex. The use of two consecutive purification steps allows for isolation of the complex, in a purified form, without disruption of the targeted complex. Only certain combinations of purification tags are suitable for this method.
The calmodulin-binding domain of the calmodulin binding peptide (CBP-tag) and the IgG binding domain(s) of Staphylococcus aureus protein A represent an efficient combination of purification tags, according to this method (Rigaut et al., supra; Puig et al., supra; U.S. 2002/0061513). The interaction between the CBP-tag and the purification matrix (immobilized calmodulin) can be controlled by the presence of Ca2+. In the presence of Ca2+, the CBP tag binds to the purification matrix whereas removal of Ca2+ with a chelating agent such as EGTA, allows recovery of the tagged protein from the purification resin under mild conditions (Stofko-Hahn et al., 1992, FEBS Lett., 302:274-278). The IgG-binding domain of protein A provides specific, high affinity binding with little non-specific interaction. However, it is very difficult to elute protein A tagged proteins from IgG-columns. Consequently, elution can only be achieved by removing protein A fusion proteins by digestion with a site-specific protease. Utilization of the IgG-binding domain of protein A therefore requires additional processing steps and leads to contamination of the purified protein with the protease.
There is a need in the art for a method to detect and identify protein complexes that does not disrupt protein-protein interactions. This method will also facilitate detection of binding partners for a protein of interest in the absence of prior knowledge of the binding partner(s) or the function of the protein complex. There is also a need in the art for a purification protocol for protein complexes that does not require digestion with a protease enzyme. This method provides a simple, generic purification protocol that can be used routinely, and, possibly, in an automated system, for the purification of protein complexes and for proteome analysis.